Synthesis of beta-hydroxyisovalerate and methods of use

ABSTRACT

The biological production of beta-hydroxyisovalerate (βHIV) using a non-natural microorganism. The non-natural microorganism for the biologically-derived βHIV provides more beta-hydroxyisovalerate synthase activity than the wild-type parent. The non-natural microorganism can host a non-natural enzyme, such as the non-natural enzyme expressed in a yeast or bacteria, wherein the non-natural microorganism comprises an active βHIV metabolic pathway for the production of βHIV. The biological derivation of βHIV eliminates toxic by-products and impurities that result from the chemical production of βHIV, such that βHIV produced by a non-natural microorganism prior to any isolation or purification process has not been in substantial contact with any halogen-containing component.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional Application No.63/171,418, filed Apr 6, 2021, which is hereby incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present disclosure generally relates to biological processes ofproducing beta hydroxyisovalerate, more particularly methods to createnon-natural microorganisms comprising non-natural βHIV synthase enzymesand processes for using said microorganisms to produce betahydroxyisovalerate, and more specifically to non-natural microorganismsthat produce beta hydroxyisovalerate.

BACKGROUND

The beta hydroxyisovalerate (βHIV) molecule (shown below), which is alsoknown as 3-hydroxy-3-methylbutric acid, has potential applicationsranging from liquid crystals to pharmaceutical ingredients and dietarysupplements.

As such, a number of methods to produce β-hydroxyisovalerate are knownin the art. They are mainly centered around chemical, organic synthesisstarting with 4-hydroxy-4-methyl-2-pentanone. βHIV can be synthesized bythe oxidation of 4-hydroxy-4-methyl-2-pentanone. One suitable procedureis described by Coffman et al., J. Am. Chem. Soc. 80: 2882-2887 (1958).See also, for example, U.S. Pat. Nos. 6,248,922, 6,090,978 US1016471653, U.S. Pat. No. 6,090,918 and US2014025698. As describedtherein, βHIV is synthesized by an alkaline sodium hypochloriteoxidation of diacetone alcohol. The product is recovered in free acidform, which can be converted to a salt. For example, βHIV can beprepared as its calcium salt by a procedure similar to that of Coffmanet al. (1958) in which the free acid of βHIV is neutralized with calciumhydroxide and recovered by crystallization from an aqueous ethanolsolution.

Biological methods to produce βHIV are also known. For example, βHIV canalso be prepared by the conversion of 3-methylcrotonate(3-methylbut-2-enoate) by cell-free extracts of Galactomyces reessii[Dhar and J P N Rosazza. Journal of Industrial Microbiology &Biotechnology 2002, 28, 81-87]. Cell free extracts of Galactomycesreessii contain an enoyl CoA hydratase that can catalyze thetransformation of 3-methylcrotonic acid to βHIV. Resting cells ofGalactomyces reessii could convert β-methylbutyrate intoβ-hydroxyisovalerate [Lee I Y, Nissen S L, Rosazza J P. Applied andenvironmental microbiology 1997, 63(11):4191-4195; Lee I Y, Rosazza J P.Arch. Microbiol., 1998 Mar;169(3):257-62]. Using a two-step fed-batchfermentation process where biomass was first produced to sufficientdensity in the first step, followed by the addition of β-methylbutyrateto the washed biomass in the second step, Lee et al. reported producing38 g/L of βHIV. U.S. Pat. No. 10,676,765B2 describes an alternativeenzymatic method to produce βHIV through the conversion of3-methylcrotonyl-CoA into βHIV via 3-hydroxy-3-methylbutyryl-CoA. Theavailability of 3-methylcrotonic acid or β-methylbutyrate ineconomically viable quantities for in vitro or in vivo production ofβHIVis still a challenge that needs to be overcome before this process canbecome commercially viable.

Indeed, βHIV is synthesized in humans through the metabolism ofL-leucine (see for example Nutrient Metabolism, Martin Kohlmeier,Academic Press, 2015) as a result of the conversion of its keto acid,α-ketoisocaproate (KIC) by the promiscuous action of4-hydroxyphenylpyruvate dioxygenase (HPPD). Dioxygenases are enzymesthat incorporate diatomic oxygen to form oxo-intermediates. To reducediatomic oxygen, these enzymes require a source of electrons as well asa cofactor capable of one-electron chemistry. The ferrous ion is themost common cofactor capable of localizing substrates by acting as aconduit to transfer the electrons from the substrates to oxygen. Commoncoordinated reductant for the ferrous ion is the α-keto acid moiety andα-keto acid dependent oxygenases are very versatile and play a key rolein the secondary metabolism [Purpero and Moran, J. Biol. Inorg. Chem. 12(2007) 587-601].

A majority of the α-keto acid dependent oxygenases have threesubstrates—oxygen, α-ketoglutarate (the source of the α-keto acid) andthe substrate, whose transformation is the catalytic objective[Hausinger, Crit. Rev. Biochem. Mol. Biol. 39 (2004) 21-68]. HPPD andhydroxymandelate synthase (HMS) are an exception to this generalprincipal by having only two substrates. HPPD and HMS receive electronsfrom their common α-keto acid substrate, 4-hydroxyphenylpyruvate (HPP),and also transform it into their hydroxylated and decarboxylatedproducts homogentisate and hydroxymandelate, respectively, without theneed for α-ketoglutarate. These two enzymes are believed to have evolvedfrom an entirely different lineage than all other α-keto acid oxygenases[Moran, G. M., Archives of Biochemistry and Biophysics 544 (2014) 58-68]although their core catalytic mechanism is consistent with the enzymefamily.

There is a large body of literature on HPPD, owing to its importance inagriculture and medicine. The primary product of HPPD reaction ishomogentisate, which is the precursor to plastoquinone and tocopherolsin plants and archaea. They are intimately involved in electrontransport in the photosynthetic system, serve as antioxidants and planthormones. Therefore, inhibiting the synthesis of homogentisate iscommonly used to inhibit the growth of plants and weeds. A number ofmolecules such as leptospermone and usnic acid and their similarsinhibit HPPD activity and are used as ingredients in herbicides[Beaudegnies et al., Bioorg. Med. Chem. 17 (2009) 4134-4152]. HPPDinhibitors such as NTBC (nitisinone) is used to treat Type 1tyrosinemia. Inborn genetic errors leading to aberrant metabolic enzymesin the catabolism of homogentisate causes Type 1 tyrosinemia. NTBC hasbeen used as a treatment by repressing the synthesis of homogentisate byinhibiting HPPD [Lindstedt et al., Lancet 340 (1992) 813-817].

Interestingly, HPPD was also shown to produce βHIV as a result of itspromiscuity towards α-ketoisocaproate, the keto acid of leucine [CrouchN P, E. Baldwin, M.-H. Lee, C. H. MacKinnon, Z. H. Zhang, Bioorg MedChem Lett 1996, 6(13):1503-1506]. In addition to its involvement inaromatic amino acid metabolism, HPPD is involved in the metabolism ofleucine by converting excess α-ketoisocaproate into βHIV [Crouch N P,Lee M H, Iturriagagoitia-Bueno T, MacKinnon C H. Methods in enzymology2000, 324:342-355]. Prior to the elucidation of the promiscuity of HPPD,a dedicated dioxygenase to transform α-ketoisocaproate into βHIV wasalleged to exist [Sabourin P J, Bieber L L: The Journal of biologicalchemistry 1982, 257(13):7468-7471; Sabourin P J, Bieber L L: Methods inenzymology 1988, 166:288-297; Sabourin P J, Bieber L L: Metabolism:clinical and experimental 1983, 32(2):160-164; Xu et al., Biochemicaland Biophysical Research Communications 276, (2000), 1080-1084]. Baldwinet al., (1995) published early reports of HPPD having several foldhigher activity with HPP than with α-ketoisocaproate [Baldwin et al.,Bioorganic and Medicinal Chemistry Letters, 5(12) (1995), 1255-1260].Subsequently, sequence studies and further biochemical analyses byCrouch et al, (1996) and Crouch et al., (2000) confirmed that thealleged dioxygenase was HPPD which catalyzed the conversion ofα-ketoisocaproate into βHIV as a result of its promiscuity. Indeed,Crouch et al., 1996 suggested any further reference to HPPD asα-ketoisocaproate dioxygenase be discontinued. The promiscuity of HPPDis also evident by its transformation of 2-keto-4-(methylthio)butyricacid, the keto acid of methionine [Adlington, R. M., et al., Bioorganic& Medicinal Chemistry Letters, Volume 6, Issue 16, 20 August 1996,2003-2006].

There are several examples in the food, pharmaceutical, animal feed,biofuel, and biopolymer industries of producing ingredients through theuse of metabolically engineered microorganisms and employing them in afermentation process. Not all microorganisms are suited for theproduction of products. For example, bacteria are conventionally bettersuited for the production of amino acids, vitamins and enzymes whileyeasts are better suited for the production of alcohols and organicacids. Therefore, selecting the appropriate microorganism to produceβHIV is critical. This disclosure relates to methods of selecting amicroorganism for βHIV production.

Given that βHIV is produced using chemical processes that are not onlyenergy-intensive, but also result in toxic by-products, there is a clearand urgent need to develop environmentally benign processes that userenewable feedstocks. There is also a need for the production of highquality βHIV that is cost-effective and efficiently produced.

SUMMARY

The subject of the present disclosure satisfies the need and providesrelated advantages as well. Provided herein are certain embodiments tocreate non-natural microorganisms to express or overexpress the βHIVmetabolic pathway, methods of making these microorganisms, and using themicroorganisms to produce βHIV.

Provided herein are methods to select and engineer microorganisms toproduce beta hydroxyisovalerate (βHIV) and uses of the engineered,non-natural microorganisms. This disclosure also provides methods ofproducing βHIV by culturing the genetically modified microorganisms inthe presence of at least one carbon source, then isolating βHIV from theculture. In certain embodiments, the carbon source is one or more ofglucose, xylose, arabinose, sucrose and lactose.

In some embodiments, a non-natural microorganism comprises a metabolicpathway relating to one or more steps of (i) pyruvate to acetolactate,(ii) acetolactate to 2,3-dihydroxyisovalerate, (iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate, (iv) α-ketoisovalerate toα-isopropylmalate, (v) α-isopropylmalate to β-isopropylmalate, (vi)β-isopropylmalate to α-ketoisocaproate and (vii) α-ketoisocaproate toβHIV. In some aspects, if the non-natural microorganism has differentcellular compartments, one or more genes for the one or more steps (i)to (vii) for the metabolic pathway encodes an enzyme that is localizedto the cytosol.

In some embodiments, a non-natural microorganism comprises a metabolicpathway relating to one or more steps of (i) pyruvate into acetolactate,(ii) acetolactate into 2,3-dihydroxyisovalerate, (iii) 2,3-dihydroxyisovalerate into α-ketoisovalerate, (iv) α-ketoisovalerateinto 2-isopropylmalate, (v) 2-isopropylmalate into 2-i sopropylmaleate,(vi) 2-i sopropylmaleate into 3-isopropylmalate, (vii) 3-isopropylmalateinto 2-isopropyl-3-oxosuccinate, (viii) 2-isopropyl-3-oxosuccinate intoα-ketoisocaproate, and (ix) α-ketoisocaproate into βHIV. In someaspects, one or more genes for the one or more steps (i) to (ix) of themetabolic pathway encodes an enzyme that is localized to the cytosol.

In some embodiments, the non-natural microorganism expresses oroverexpresses at least one of the genes encoding for acetolactatesynthase, keto-acid reductoisomerase, dihydroxyacid dehydratase,2-isopropylmalate synthase, isopropylmalate isomerase, 3-isopropylmalatedehydrogenase and βHIV synthase. In some aspects, the non-naturalmicroorganism expresses or overexpresses two or more genes encoding foracetolactate synthase, keto-acid reductoisomerase, dihydroxyaciddehydratase, 2-isopropylmalate synthase, isopropylmalate isomerase,3-isopropylmalate dehydrogenase and βHIV synthase.

In certain embodiments, the non-natural microorganisms havingcompartmentalized metabolism comprise a βHIV producing metabolic pathwaywith at least one βHIV pathway enzyme localized in the cytosol. In anexemplary embodiment, the non-natural microorganisms comprise a βHIVproducing metabolic pathway with all the βHIV pathway enzymes localizedin the cytosol.

In some embodiments, the non-natural eukaryotic microorganism expressesor overexpresses at least one of the genes encoding for cytosolicacetolactate synthase, cytosolic keto-acid reductoisomerase, cytosolicdihydroxyacid dehydratase, cytosolic 2-isopropylmalate synthase,cytosolic isopropylmalate isomerase, cytosolic 3-isopropylmalatedehydrogenase and cytosolic βHIV synthase. In some aspects, thenon-natural eukaryotic microorganism expresses or overexpresses two ormore genes encoding for cytosolic acetolactate synthase, cytosolicketo-acid reductoisomerase, cytosolic dihydroxyacid dehydratase,cytosolic 2-isopropylmalate synthase, cytosolic isopropylmalateisomerase, cytosolic 3-isopropylmalate dehydrogenase and cytosolic βHIVsynthase.

In some embodiments, the non-natural microorganism comprises at leastone nucleic acid encoding a polypeptide with beta hydroxyisovaleratesynthase activity wherein said polypeptide is at least about 65%identical to at least one polypeptide selected from SEQ ID NOs: 1-3. Incertain embodiments, the polypeptide with βHIV synthase activity isderived from Rattus norvegicus.

In certain embodiments, the non-natural microorganism comprises at leastone nucleic acid encoding a polypeptide with βHIV synthase activitywherein said polypeptide is at least about 65% identical to at least onepolypeptide selected from SEQ ID NOs: 4-5. In certain embodiments, thepolypeptide with βHIV synthase activity is derived from Yarrowialipolytica. In certain embodiments, the non-natural microorganismcomprises at least one nucleic acid encoding a polypeptide with βHIVsynthase activity wherein said polypeptide is at least about 65%identical to at least one polypeptide selected from SEQ ID NOs: 6-8. Incertain embodiments, the polypeptide with βHIV synthase activity isderived from Homo sapiens.

In another embodiment, the non-natural microorganism comprises adioxygenase enzyme which has been modified or mutated to increase theability of the enzyme to preferentially utilize α-ketoisocaproate as itssubstrate. According to certain aspects of the present invention, thenon-natural enzyme comprises one or more dioxygenase enzymes having oneor more modifications or mutations at substrate-specificity positionscorresponding to amino acids selected from A361, F336, F347, F364, F368,F371, G362, 1227, 1252, L224, L289, L323, L367, N187, N241, N363, P239,Q251, Q265, 5226, V212, V217, V228 and W210 of SEQ ID NO: 1.

In some aspects, at least one of the substrate-specificity positionscorresponding to amino acids selected from the group consisting of A361,F336, F347, F364, F368, F371, G362, I227, I252, L224, L289, L323, L367,N187, N241, N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 ofSEQ ID NO: 1 has been replaced with one of the corresponding disclosedamino acids to alter the respective substrate-specificity residue.

In some other aspects, two or more of the substrate-specificitypositions corresponding to amino acids selected from the groupconsisting of A361, F336, F347, F364, F368, F371, G362, I227, I252,L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212,V217, V228 and W210 of SEQ ID NO: 1 have been replaced with one of thecorresponding disclosed amino acids to alter the respectivesubstrate-specificity residue.

In yet some other aspects, at least 3 and up to 24 of thesubstrate-specificity positions corresponding to amino acids selectedfrom the group consisting of A361, F336, F347, F364, F368, F371, G362,1227, 1252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265,S226, V212, V217, V228 and W210 of SEQ ID NO: 1 have been replaced withone of the corresponding disclosed amino acids to alter the respectivesubstrate-specificity residue.

According to certain aspects of the present invention, the non-naturalenzyme comprises one or more modifications at substrate-specificitypositions corresponding to amino acids selected from A361, F336, F347,F364, F368, F371, G362, I227, I252, L224, L289, L323, L367, N187, N241,N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 of SEQ ID NO: 6.

In some aspects, at least one of the substrate-specificity positionscorresponding to amino acids selected from the group consisting of A361,F336, F347, F364, F368, F371, G362, I227, I252, L224, L289, L323, L367,N187, N241, N363, P239, Q251, Q265, S226, V212, V217, V228 and W210 ofSEQ ID NO: 6 has been replaced with one of the corresponding disclosedamino acids to alter the respective substrate-specificity residue.

In some other aspects, two or more of the substrate-specificitypositions corresponding to amino acids selected from the groupconsisting of A361, F336, F347, F364, F368, F371, G362, I227, I252,L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212,V217, V228 and W210 of SEQ ID NO: 6 have been replaced with one of thecorresponding disclosed amino acids to alter the respectivesubstrate-specificity residue.

In yet some other aspects, at least 3 and up to 24 of thesubstrate-specificity positions corresponding to amino acids selectedfrom the group consisting of A361, F336, F347, F364, F368, F371, G362,I227, I252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265,S226, V212, V217, V228 and W210 of SEQ ID NO: 6 have been replaced withone of the corresponding disclosed amino acids to alter the respectivesubstrate-specificity residue.

In certain embodiments, the non-natural microorganisms may beprokaryotic microorganisms. In another embodiment, the non-naturalmicroorganism may be an eukaryotic microorganism. In certainembodiments, the non-natural eukaryotic microorganisms may benon-natural yeast microorganisms. In some embodiments, the non-naturalyeast may be Crabtree-negative yeasts. In some embodiments, thenon-natural yeast microorganism may be selected from the groupconsisting of Saccharomyces, Kluyveromyces, Pichia, Issatchenkia,Hansenula, or Candida.

In another embodiment, the non-natural microorganism may be cultivatedin a culture medium containing a feedstock providing the carbon sourceuntil a recoverable quantity ofβHIV is produced and optionally,recovering the βHIV. In certain embodiments, the non-naturalmicroorganism produces βHIV from a carbon source with a yield of atleast about 0.1 percent of theoretical yield. In another aspect, thenon-natural microorganism produces βHIV from a carbon source with ayield of at least 1 percent of theoretical yield. In another aspect, thenon-natural microorganism produces βHIV from a carbon source with ayield of at least about 5 percent of theoretical yield. In anotheraspect, the non-natural microorganism produces βHIV from a carbon sourcewith a yield of at least 20 percent of theoretical yield. In anotheraspect, the non-natural microorganism produces βHIV from a carbon sourcewith a yield of at least 50 percent, at least about 75 percent, at leastabout 80 percent, or at least about 85 percent of the theoretical yield.

In some aspects, the non-natural microorganism produces βHIV from acarbon source with a yield of at least about 0.1 percent up to 100percent of theoretical yield, in some aspects at least about 1 percentup to 99.9 percent of theoretical yield, in some aspects at least about5 percent up to about 99.5 of theoretical yield, in some aspects atleast 20 percent up to about 99.5 percent of theoretical yield, in someaspects at least 50 percent up to about 99.5 percent of theoreticalyield, in some aspects at least about 75 percent up to about 99.5percent of theoretical yield, in some aspects at least about 80 percentup to about 99.5 percent of theoretical yield, and in some aspects atleast about 85 percent up to about 99.5 percent of theoretical yield.

In some embodiments, the present invention is directed to a compositioncomprising βHIV produced by a non-natural microorganism, wherein theβHIV prior to any isolation or purification process has not been insubstantial contact with any component comprising a halogen-containingcomponent. In some aspects, the halogen-containing component is achemical derivative produced by a typical chemical production process ofβHIV. In some aspects, the halogen-containing component compriseshydrochloric acid and/or chloroform.

The above summary is not intended to describe each illustratedembodiment or every implementation of the subject matter hereof. Thefigures and the detailed description that follow more particularlyexemplify various embodiments.

BRIEF DESCRIPTION OF THE FIGURES

Subject matter hereof may be more completely understood in considerationof the following detailed description of various embodiments inconnection with the accompanying figures, in which:

FIG. 1 illustrates a βHIV metabolic pathway, according to certainembodiments of the present invention. According to some aspects thisdisclosure, the metabolic pathway can also comprise an activetransporter to transport βHIV out of the non-natural microorganism.

FIG. 2 illustrates another βHIV metabolic pathway, according to certainembodiments of the present invention. According to some aspects of thisdisclosure, the metabolic pathway can also comprise an activetransporter to transport βHIV out of the non-natural microorganism.

FIG. 3 illustrates the growth of microorganisms as maximum specificgrowth rate in the presence of various concentrations ofβHIV in themedia as an indicator of metabolic activity.

FIG. 4 illustrates the production ofβHIV using non-natural bacteria.

FIG. 5 is a bar graph illustrating the accumulation of βHIV bynon-natural yeast microorganism harboring an unmodified dioxygenaseenzyme (SB556) or an improved βHIV synthase (SB557), according tocertain embodiments of the present invention.

While various embodiments are amenable to various modifications andalternative forms, specifics thereof have been shown by way of examplein the drawings and will be described in detail. It should beunderstood, however, that the intention is not to limit the claimedinventions to the particular embodiments described. On the contrary, theintention is to cover all modifications, equivalents, and alternativesfalling within the spirit and scope of the subject matter as defined bythe claims.

DETAILED DESCRIPTION OF THE INVENTION

The following description of the present disclosure is merely intendedto illustrate various embodiments. As such, the specific modificationsdiscussed are not to be construed as limitations on the scope of thepresent disclosure. It will be apparent to one skilled in the art thatvarious equivalents, changes, and modifications may be made withoutdeparting from the scope of the present disclosure, and it is understoodthat such equivalent embodiments are to be included herein. Allreferences cited herein are incorporated by reference in their entirety.

The present disclosure relates to non-natural microorganisms and the useof said microorganisms in a fermentation process to produce higher valueproducts such as organic acids. More specifically, the presentdisclosure relates to engineered microorganisms that produceβ-hydroxyisovaleric acid (βHIV). As a molecule with unique structure,βHIV has potential applications ranging from liquid crystals topharmaceutical ingredients and dietary supplements.

As used herein, “β-hydroxyisovalerate” or “beta hydroxyisovalerate” or“βHIV” or “β-hydroxy-β-methylbutyrate” or “3-hydroxy-3-methylbutyricacid” refer to the same compound having the following molecularstructures (free acid form on left and conjugate base on the right).

Furthermore, these terms not only include the free acid form orconjugate base, but also the salt form with a cation and derivativesthereof, or any combination of these compounds. For instance, a calciumsalt ofβHIV includes calcium βHIV hydrate having the following molecularstructure.

While the foregoing terms mean any form ofβHIV, the form ofβHIV usedwithin the context of the present disclosure preferably is selected fromthe group comprising of a free acid, a calcium salt, an ester and alactone.

As used herein, the term “microorganism” refers to a prokaryote such asa bacterium or a eukaryote such as a yeast or a fungus. As used herein,the term “non-natural microorganism” refers to a microorganism that hasat least one genetic alteration not normally found in a naturallyoccurring strain of the species, including wild-type strains of thereference species. Genetic alterations include, for example,human-intervened modifications introducing expressible nucleic acidsencoding polypeptides, other nucleic acid additions, nucleic aciddeletions and/or other functional disruption of the microorganism'sgenetic material. When a microorganism is genetically engineered tooverexpress a given enzyme, it is manipulated such that the host cellhas the capability to express, and preferably, overexpress an enzyme,thereby increasing the biocatalytic capability of the cell. When amicroorganism is engineered to inactivate a gene, it is manipulated suchthat the host cell has decreased, and preferably, lost the capability toexpress an enzyme. As used herein, the term “overexpress” refers toincreasing the expression of an enzyme to a level greater than the cellnormally produces. The term encompasses overexpression of endogenous aswell as exogenous enzymes. As used herein, the terms “gene deletion” or“gene knockout” or “gene disruption” refer to the targeted disruption ofthe gene in vivo resulting in the removal of one or more nucleotidesfrom the genome resulting in decreased or loss of function using geneticmanipulation methods such as homologous recombination, directedmutagenesis or directed evolution.

As used herein, the term “gene” refers to a nucleic acid sequence thatcan be transcribed into messenger RNA and further translated intoprotein.

The term “nucleic acid” as used herein, includes reference to adeoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, ineither single- or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e.g., peptide nucleicacids). A polynucleotide can be full-length or a subsequence of a nativeor heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof.

Usually, the nucleotide sequence encoding an enzyme is operably linkedto a promoter that causes sufficient expression of the correspondingnucleotide sequence in the host microorganism according to the presentdisclosure to confer to the cell the ability to produceβ-hydroxyisovaleric acid. As used herein, the term “operably linked”refers to a linkage of polynucleotide elements (or coding sequences ornucleic acid sequence) in a functional relationship. A nucleic acidsequence is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the coding sequence. In order to increasethe likelihood that an exogenous gene is translated into an enzyme thatis in active form, the corresponding nucleotide sequence may be adaptedto optimize its codon usage to that of the chosen host microorganism.Several methods for codon optimization are known in the art and areembedded in computer programs such as CodonW, GenSmart, CodonOpt, etc.

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for a nucleic acidpolymerase, transcription initiation sites and any other DNA sequencesknown to one of skill in the art. A “constitutive” promoter is apromoter that is active under most environmental and developmentalconditions. An “inducible” promoter is a promoter that is active underenvironmental or developmental regulation.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, the protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation.

The term “enzyme” as used herein is defined as a protein which catalyzesa (bio)chemical reaction in a cell. The interaction of an enzyme withother molecules such as the substrate can be quantified by the Michaelisconstant (K_(M)), which indicates the affinity of the substrate to theactive site of the enzyme. K_(M) can be quantified using prior art (seefor example, Stryer, Biochemistry, 4th edition, W. H. Freeman, Nelsonand Cox, Lehninger Principles of Biochemistry, 6th edition, W. H.Freeman). The rate of biocatalysis or enzymatic activity is defined byk_(cat), which is the enzyme turnover number. Therefore, the ratio ofthe rate of enzymatic activity to the substrate affinity is widelyconsidered to be representative of an enzyme's catalytic efficiency. Asdefined herein, the efficiency of an enzyme to act on a specificsubstrate is quantified by the ratio of k_(cat)/K_(M). Therefore, anenzyme with higher value of k_(cat)/K_(M) for a certain substrate cancatalyze the reaction more efficiently than another enzyme with a lowervalue of k_(cat)/K_(M) for the same substrate. A non-natural enzymerefers to an enzyme that comprises at least one amino acid alteration atthe desired position that is not normally found in nature. Amino acidalternations include, for example, human-intervened modificationsintroducing replacing one naturally occurring amino acid with another,addition or deletion of amino acids such that the modified enzyme hasthe capability of enhanced catalytic activity.

As used herein, β-hydroxyisovalerate synthase refers to an enzyme thatcan catalyze the conversion of α-ketoisocaproate into βHIV. One Unit (U)of βHIV synthase activity is defined here as the amount of enzyme neededto convert one micromole of α-ketoisocaproate into βHIV in one minuteunder the reaction conditions. Accordingly, a variant of βHIV synthasethat can convert more α-ketoisocaproate into βHIV than the same amountof another variant is preferred.

The term “biosynthetic pathway”, also referred to as “metabolicpathway”, refers to a set of anabolic or catabolic biochemical reactionsfor converting one chemical species into another. Gene products belongto the same “metabolic pathway” if they, in parallel or in series, acton the same substrate, produce the same product, or act on or produce ametabolic intermediate (i.e., metabolite) between the same substrate andmetabolite end product. As used herein, the term “βHIV metabolicpathway” or “βHIV pathway” refers to an enzyme pathway which producesβHIV from pyruvate, as illustrated in FIGS. 1 or FIG. 2.

The present disclosure relates to a non-natural microorganism forproducing βHIV. Tolerance to high concentrations of βHIV is an importanttrait of a suitable microorganism. An ideal microorganism to enable βHIVproduction is capable of conducting fermentation at low pH levels todecrease downstream recovery costs, resulting in more economicalproduction. Additional characteristics of a suitable microorganisminclude rapid growth and exhibit overall process robustness.

In some embodiments, the subject of the present disclosure relates to anon-natural microorganism having an active βHIV metabolic pathway frompyruvate to βHIV.

A βHIV metabolic pathway is shown in FIG. 1. In some embodiments, βHIVmetabolic pathway comprises of the conversion of pyruvate into2-acetolactate, 2-acetolactate into 2,3-dihydroxy-isovalerate,2,3-dihydroxy-isovalerate into α-ketoisovalerate, α-ketoisovalerate into2-isopropylmalate, 2-isopropylmalate into 3-isopropylmalate,3-isopropylmalate into KIC and KIC into βHIV.

Another βHIV metabolic pathway is shown in FIG. 2. In some embodiments,βHIV metabolic pathway comprises of the conversion of pyruvate into2-acetolactate, 2-acetolactate into 2,3-dihydroxy-isovalerate,2,3-dihydroxy-isovalerate into α-ketoisovalerate, α-ketoisovalerate into2-i sopropylmalate, 2-isopropylmalate into 2-i sopropylmaleate,2-isopropylmaleate into 3-i sopropylmalate, 3-i sopropylmalate into2-isopropyl-3-oxosuccinate, 2-isopropyl-3-oxosuccinate into KIC, KICinto βHIV.

In some embodiments, the βHIV pathway also comprises a hydroxy acidtransporter to facilitate the export ofβHIV formed inside themicroorganism to extracellular environment.

As used herein, the “theoretical yield” of βHIV refers to the molarratio of βHIV that is produced extracellularly to the carbon source thatis used. The theoretical yield can be calculated based on thebiochemical conversion of glucose to pyruvate and the subsequentconversion of pyruvate into βHIV in the βHIV metabolic pathway, takinginto consideration of the microorganism's native redox constraints toenable the conversion. For example, in yeast, the theoretical yieldofβHIV from glucose is 0.667.

In a first example embodiment, the non-natural microorganism of thepresent disclosure (a) expresses or overexpresses at least one geneencoding for βHIV synthase, (b) expresses or overexpresses at least onegene encoding for acetolactate synthase, (c) expresses or overexpressesat least one gene encoding for acetohydroxy acid reductoisomerase, (d)expresses or overexpresses at least one gene encoding for 2,3-dihydroxyisovalerate dehydratase, (e) expresses or overexpresses at least onegene encoding for 2-isopropylmalate synthase (f) expresses oroverexpresses at least one gene encoding for isopropylmalate isomerase,or (g) expresses or overexpresses at least one gene encoding for3-isopropylmalate dehydrogenase. In some aspects, the non-naturalmicroorganism of the present disclosure comprises a combination of twoor more of (a), (b), (c) (d), (e), (f) and (g).

In a second example embodiment, the non-natural microorganism of thepresent disclosure (a) expresses or overexpresses at least one geneencoding for βHIV synthase, (b) expresses or overexpresses at least onegene encoding for acetolactate synthase, (c) expresses or overexpressesat least one gene encoding for 2,3-keto-acid reductoisomerase, (d)expresses or overexpresses at least one gene encoding for dihydroxyisovalerate dehydratase, (e) expresses or overexpresses at least onegene encoding for 2-isopropylmalate synthase, (i) expresses oroverexpresses at least one gene encoding for 2-isopropylmalatehydrolyase (2-isopropylmaleate-forming), (j) expresses or overexpressesat least one gene encoding for 2-isopropylmalate hydrolyase(3-isopropylmalate-forming), (g) expresses or overexpresses at least onegene encoding for 3-isopropylmalate dehydrogenase. In some aspects, thenon-natural organism of the present disclosure comprises a combinationof two or more of (a), (b), (c) (d), (e), (i), (j) and (g).

In some embodiments, the non-natural microorganism of the presentdisclosure expresses or overexpresses at least one gene encoding apolypeptide with acetolactate synthase (EC: 2.2.1.6) activity. Forexample, acetolactate synthases capable of converting pyruvate toacetolactate may be derived from a variety of sources (e.g., bacterial,yeast, Archaea, etc.), including B. subtilis (GenBank Accession No.Q04789.3), L. lactis (GenBank Accession No. NP_267340.1), S. mutans(GenBank Accession No. NP_721805.1), K. pneumoniae (GenBank AccessionNo. PTD93137.1), C. glutamicum (GenBank Accession No. 1238373540), E,cloacae (GenBank Accession No. WP_013097652.1), M. maripaiudis (GenBankAccession No. ABX01060.1), P. grisea (GenBank Accession No. AAB81248.1),T. stipitatus (GenBank Accession No. XP_002485976.1), or S. cerevisiaeILV2 (GenBank Accession No. 1789111829). Additional acetolactatesynthases capable of converting pyruvate to acetolactate are describedin WO2013016724, which incorporated herein by reference in its entirety.A review article characterizing the biosynthesis of acetolactate frompyruvate via the activity of acetolactate synthases is provided byChipman et al., 1998, Biochimica et Biophysica Acta 1385: 401-19.Chipman et al, provide an alignment and consensus for the sequences of arepresentative number of acetolactate synthases. Motifs shared in commonbetween the majority of acetolactate synthases include:SGPG(A/C/V)(T/S)N, GX(P/A)GX(V7A/T),GX(Q/G)(T/A)(IJM)G(Y/F/W)(A/G)X(P/G)(W/A)AX(G/T)(A/V) andGD(G/A)(G/S/C)F, at amino acid positions corresponding to the 163-169,240-245, 521-535, and 549-553 residues, respectively, of the S.cerevisiae ILV2. Thus, a protein harboring one or more of these aminoacid motifs can generally be expected to exhibit acetolactate synthaseactivity. In some embodiments, the non-natural microorganism of thepresent disclosure expresses or overexpresses at least one gene encodinga polypeptide that is at least about 65% identical to at least onepolypeptide selected from SEQ ID NOs: 297-300.

In some embodiments, the non-natural microorganism of the presentdisclosure expresses or overexpresses at least one gene encoding apolypeptide with acetohydroxy acid reductoisomerase activity (EC:1.1.1.86). Acetohydroxy acid reductoisomerases capable of convertingacetolactate to 2,3-dihydroxyisovaierate may be derived from a varietyof sources (e.g., bacterial, yeast, Archaea, etc.), including E. coli(GenBank Accession No. EGB30597.1), L. lactis (GenBank Accession No.WP_012897822.1), Shewanella sp, (GenBank Accession No. WP_011621167.1),A. fischeri (GenBank Accession No. WP_005421503.1), M. maripaludis(GenBank Accession No. ABO35228.1), B. subtilis (GenBank Accession No.CAB14789), S. pombe (GenBank Accession No. NP_001018845) or S.cerevisiae ILV5 (GenBank Accession No. NP_013459.1). Additionalketol-acid reductoisomerases capable of converting acetolactate to2,3-dihydroxyisovalerate are described in WO2013016724, incorporatedherein by reference in its entirety. Motifs shared between a majority ofacetohydroxy acid reductoisomerases include G(Y/C/W)GXQ(G/A),(F/Y/L)(S/A)HG(F/L), V(V/I/F)(M/L/A)(A/C)PK, D(L/I)XGE(Q/R)XXLXG andS(D/NAT)TA(E/Q/R)XG at amino acid positions corresponding to the 89-94,175-179, 194-200, 282-272, and 459-465 residues, respectively, of the E.coli acetohydroxy acid reductoisomerase encoded by ilvC. Thus, a proteinharboring one or more of these amino acid motifs can generally beexpected to exhibit acetohydroxy acid reductoisomerase activity. Thenaturally existing acetohydroxy acid reductoisomerases preferentiallyuse NADPH as a cofactor. Cofactor specificity can be switched topreferentially use NADH as a cofactor by means of modifying specificresidues. Examples of such acetohydroxy acid reductoisomerases withincreased preference for using NADH as a cofactor are described in USPublication No. 2010/0143997. In some embodiments, the non-naturalmicroorganism of the present disclosure expresses or overexpresses atleast one gene encoding a polypeptide that is at least about 65%identical to at least one polypeptide selected from SEQ ID NOs: 301-303.

In some embodiments, the non-natural microorganism of the presentdisclosure expresses or overexpresses at least one gene encoding apolypeptide with 2,3-dihydroxy isovalerate dehydratase activity (EC:4.2.1.9). Dihydroxy acid dehydratases capable of converting2,3-dihydroxyisovalerate to α-ketoisovalerate may be derived from avariety of sources (e.g., bacterial, yeast, Archaea, etc.), including M.tuberculosis (GenBank Accession No. CLR57443), L. lactis (GenBankAccession No. WP_010905837.1), S. mutans (GenBank Accession No.WP_002262431.1), M. stadtmanae (GenBank Accession No. WP_011407142.1),M. tractuosa (GenBank Accession No. WP_013453775.1), Eubacterium SCB49(GenBank Accession No. WP_118518751.1), Y. lipolytica (GenBank AccessionNo. QNP96049.1), N. crassa (GenBank Accession No. XP 963045.1), or S.cerevissae ILV3 (GenBank Accession No. NP_012550.1). Additionaldihydroxy acid dehydratases capable of 2,3-dihydroxyisovaierate toa-ketoisovalerate are described in WO02013016724, incorporated herein byreference in its entirety. Motifs shared in common between the majorityof 2,3-dihydroxy isovalerate dehydratases include: SLXSRXXIA, CDKXXPG,GXCXGXXTAN, GGSTN, GPXGXPGMRXE, ALXTDGRXSG, and GHXXPEA motifs at aminoacid positions corresponding to the 93-101, 122-128, 193-202, 276-280,482-491, 509-518, and 526-532 residues, respectively, of the E. coli2,3-dihydroxy isovalerate dehydratase. Thus, a protein harboring one ormore of these amino acid motifs can generally be expected to exhibit2,3-dihydroxy isovalerate dehydratase activity. In some embodiments, thenon-natural microorganism of the present disclosure expresses oroverexpresses at least one gene encoding a polypeptide that is at leastabout 65% identical to at least one polypeptide selected from SEQ IDNOs: 304-307.

In some embodiments, the non-natural microorganism of the presentdisclosure expresses or overexpresses at least one gene encoding apolypeptide with 2-isopropylmalate synthase activity (EC: 2.3.3.13).2-isopropylmalate synthases capable of converting3-methyl-2-oxobutanoate to (2S)-2-isopropylmalate may be derived from avariety of sources (e.g., bacterial, yeast, Archaea, etc.), including C.glutamicum (GenBank Accession No. WP_015439406), E. coli (GenBankAccession No. WP_000082850.1), S. cerevisiae (GenBank Accession No.NP_014295.1 (Leu4) and NP_014751.1 (Leu9), M. maripaludis (GenBankAccession No. WP_011171007.1) or N. crassa (GenBank Accession No.XP_964875.1). Motifs shared in common between the majority of the2-isopropylmalate synthases include: LRDGXQ, IEVXFPXXSXXD,ISXHXHNDXGXXV, AGAXXVEG, GXGERXGNXXL at amino acid positionscorresponding to the 12-17, 43-54, 199-211, 220-227, 231-241 residues,respectively, of the E. coli 2-isopropylmalate synthase. Thus, a proteinharboring one or more of these amino acid motifs can generally beexpected to exhibit 2-isopropylmalate synthase activity. In someembodiments, the non-natural microorganism of the present disclosureexpresses or overexpresses at least one gene encoding a polypeptide thatis at least about 65% identical to at least one polypeptide selectedfrom SEQ ID NOs: 308-313.

In some embodiments, the non-natural microorganism of the presentdisclosure expresses or overexpresses at least one gene encoding apolypeptide with 2-isopropylmalate isomerase activity (EC: 4.2.1.33). Insome embodiments, the isomerization of 2-isopropylmalate into3-isopropylmalate is catalyzed by an enzyme that is expressed by onegene. Such 2-isopropylmalate isomerases capable of converting2-isopropylmalate into 3-isopropylmalate may be derived from a varietyof sources, including S. cerevisiae (GenBank Accession NP_011506.1), P.kudriavzevii (GenBank Accession No. XP_029320833.1) or C. albicans(GenBank Accession No. XP_718655.1). In some embodiments, theisomerization of 2-isopropylmalate into 3-isopropylmalate is catalyzedby an enzyme that is expressed by two genes, each gene encoding for adifferent subunit. Such 2-isopropylmalate isomerases capable ofconverting 2-isopropylmalate into 3-isopropylmalate may be derived froma variety of sources (e.g., bacterial, Archaea, etc.), including M.tuberculosis (GenBank Accession No. NP_217504.1), L. lactis (GenBankAccession No. WP_095586897.1), S. mutans (GenBank Accession No.WP_002262706.1), C. glutamicum (GenBank Accession No. WP_003858858.1),M. maripaludis (GenBank Accession No. WP_011171424.1) and E. coli.MG1655 (GenBank Accession No. NP_414614.1). In some embodiments, thenon-natural microorganism of the present disclosure expresses oroverexpresses at least one gene encoding a polypeptide with2-isopropylmalate isomerase activity (EC: 4.2.1.33), containing asubunit with 3-isopropylmalate dehydratase activity. Motifs shared incommon between the majority of the enzymes include: HEVTSPQAF,DSHTXTHGAFG, AFGIGT SEVEHVXATQT, CNMXIEXGA, VFXGSCTNXRXXDL,EXCASTSNRNFEGRQG, and GHXXPEA motifs at amino acid positionscorresponding to the 33-41, 128-138, 141-157, 220-228, 342-355, and422-437, residues, respectively, of the E. coli 3-isopropylmalatedehydratase. Thus, a protein harboring one or more of these amino acidmotifs can generally be expected to exhibit 3-isopropylmalatedehydratase activity. In some embodiments, the non-natural microorganismof the present disclosure expresses or overexpresses at least one geneencoding a polypeptide that is at least about 65% identical to at leastone polypeptide selected from SEQ ID NOs: 314-315.

In some embodiments, the non-natural microorganism of the presentdisclosure expresses or overexpresses at least one gene encoding apolypeptide with 3-isopropylmalate dehydrogenase activity (EC:1.1.1.85). 3-isopropylmalate dehydrogenase capable of converting(2R,3S)-3-isopropylmalate to 4-Methyl-2-oxopentanoate may be derivedfrom a variety of sources (e.g., bacterial, yeast, Archaea, plant,etc.), including A. thahana (GenBank Accession No. NP_001322636.1), L.lactis (GenBank Accession No. WP_095586896.1), S. mutans (GenBankAccession No. WP_002262707.1), C. glutamicum (GenBank Accession No.WP_011014258.1), M. maripaludis (GenBank Accession No. WP_011170483.1),E. coli. MG1655 (GenBank Accession No. NP_414615.4), P. kudriavzevii(GenBank Accession No. XP_029322355.1), C. albicans (GenBank AccessionNo. XP_720371.1), or S. cerevisiae S288C (GenBank Accession No.NP_009911.2). Motifs shared in common between the majority of3-isopropylmalate hydratases include: DAXLLGAXGXP, VRELXGGIYFG, DKXNVL,TXNXFGDILSDEA, LXEPXHGSAPD, and NPXAXILSXAMXL motifs at amino acidpositions corresponding to the 69-79, 137-147, 260-265, 245-257,279-289, and 297-309 residues, respectively, of the E. coli3-isopropylmalate dehydrogenase. Thus, a protein harboring one or moreof these amino acid motifs can generally be expected to exhibit3-isopropylmalate dehydrogenase activity. In some embodiments, thenon-natural microorganism of the present disclosure expresses oroverexpresses at least one gene encoding a polypeptide that is at leastabout 65% identical to at least one polypeptide selected from SEQ IDNOs: 316-320.

In some embodiments, the non-natural microorganism of the presentdisclosure expresses or overexpresses at least one gene encoding apolypeptide with βHIV synthase activity. The non-natural enzymesdisclosed herein have low activity using 4-hydroxyphenylpyruvate,thereby not introducing any undesirable alterations in the metabolism.The present disclosure describes methods of increasing βHIV productionthrough the use of non-natural microorganisms. Accordingly, the presentdisclosure is directed to an isolated nucleic acid encoding apolypeptide with βHIV synthase activity, wherein the polypeptidesequence is at least 65% identical to at least one polypeptide selectedfrom any of SEQ ID Nos: 1-148. Methods to determine identity andsimilarity are codified in publicly available computer programs. Examplecomputer program methods to determine identity and similarity betweentwo sequences include BLASTP and BLASTN, publicly available from NCBIand other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894). Example parameters for amino acid sequencescomparison using BLASTP are gap open 11.0, gap extend 1, Blosum 62matrix.

In certain embodiments, the polypeptide with βHIV synthase activity isderived from the genus Rattus. In an example embodiment, the polypeptidewith βHIV synthase activity is derived from Rattus norvegicus, Falloantigen Rattus norvegicus, Rattus or Rattus losea. In anotherexample embodiment, the polypeptide with βHIV synthase activity isselected from at least one of SEQ ID NOS: 1-3.

In some embodiments, the polypeptide with βHIV synthase activity has atleast 65% identity to at least one polypeptide selected from any of SEQID NOS: 1-148. Further within the scope of the present application arepolypeptides with βHIV synthase activity which are at least about 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 97%, 98%, 99%, or99.5% identical to at least one polypeptide selected from any of SEQ IDNOS: 1-148. In some embodiments, the non-natural microorganism expressesor overexpresses a nucleic acid encoding at least one polypeptide withβHIV synthase activity selected from any of SEQ ID NOS: 149-288.

The promiscuous activity of HPPD with KIC is indicative of a basal levelrecognition of the desired substrate and the present disclosurediscloses methods to increase KIC/HPP activity by modifying certainamino acids at specific positions in the sequence. Modifying amino acidsthat play a role in the catalysis can lead to alterations in the enzymeactivity. One skilled in the art can recognize the position of theseamino acids in homologous protein sequences by aligning the sequences.Two sequences are said to be “optimally aligned” when they are alignedfor similarity scoring using a defined amino acid substitution matrix(e.g., BLOSUM62), gap existence penalty and gap extension penalty so asto arrive at the highest score possible for that pair of sequences.Amino acid substitution matrices and their use in quantifying thesimilarity between two sequences are well-known in the art. The BLOSUM82matrix is often used as a default scoring substitution matrix insequence alignment protocols such as Gapped BLAST 2.0. The gap existencepenalty is imposed for the introduction of a single amino acid gap inone of the aligned sequences, and the gap extension penalty is imposedfor each additional empty amino acid position inserted into an alreadyopened gap. The alignment is defined by the amino acids positions ofeach sequence at which the alignment begins and ends, and optionally bythe insertion of a gap or multiple gaps in one or both sequences, so asto arrive at the highest possible score. While optimal alignment andscoring can be accomplished manually, the process is facilitated by theuse of a computer-implemented alignment algorithm, e.g., gapped BLAST2.0, described in Altschul et al, (1997) Nucleic Acids Res.25:3389-3402, and made available to the public at the National Centerfor Biotechnology Information Website. Optimal alignments, includingmultiple alignments, can be prepared using, e.g., PSI-BLAST with nocompositional adjustments.

As described herein, the present inventors identified polypeptides withβHIV synthase activity. One desirable feature of a polypeptide with βHIVsynthase activity is the ability to exhibit high activity for theconversion of KIC into βHIV in the βHIV metabolic pathway. Anotherdesirable property of a polypeptide with βHIV synthase activity is thelow activity with HPP, thereby reducing the impact on other aspects ofmetabolism. The present disclosure identifies several beneficialmodifications or mutations which can be made to an existing dioxygenaseenzyme to improve the dioxygenase enzyme's ability to catalyze theconversion of KIC to βHIV with higher activity. In some embodiments, thenon-natural microorganism expresses or overexpresses at least one geneencoding a polypeptide with increased KIC/HPP activity, wherein thesequence of the polypeptide has at least one modification.

According to certain aspects of the present invention, the non-naturalenzyme comprises one or more modifications at substrate-specificitypositions corresponding to amino acids selected from A361, F336, F347,F364, F368, F371, G362, I227, I252, L224, L289, L323, L367, N187, N241,N363, P239, Q251, Q265, 5226, V212, V217, V228 and W210 of SEQ ID NO: 1.

According to certain aspects of the present invention, the non-naturalenzyme comprises one or more modifications at substrate-specificitypositions corresponding to amino acids selected from A361, F336, F347,F364, F368, F371, G362, I227, I252, L224, L289, L323, L367, N187, N241,N363, P239, Q251, Q265, 5226, V212, V217, V228 and W210 of SEQ ID NO: 6.

In some embodiments, the dioxygenase enzyme has been modified or mutatedto alter one or more substrate-specificity residues. In certainembodiments, the dioxygenase enzyme is modified, wherein the residuecorresponding to position 361 of SEQ ID NO: 1 is replaced with a residueselected from methionine, leucine and isoleucine. In another embodiment,the dioxygenase enzyme is modified, wherein the residue corresponding toposition 336 of SEQ ID NO: 1 is replaced with leucine, methionine,isoleucine and tryptophan. In another embodiment, the dioxygenase enzymeis modified, wherein the residue corresponding to position 347 of SEQ IDNO: 1 is replaced with tryptophan, tyrosine and isoleucine. In anotherembodiment, the dioxygenase enzyme is modified, wherein the residuecorresponding to position 364 of SEQ ID NO: 1 is replaced withmethionine, alanine, isoleucine, leucine and tryptophan. In anotherembodiment, the dioxygenase enzyme is modified, wherein the residuecorresponding to position 368 of SEQ ID NO: 1 is replaced with tyrosine,tryptophan, leucine, isoleucine and methionine. In another embodiment,the dioxygenase enzyme is modified, wherein the residue corresponding toposition 371 of SEQ ID NO: 1 is replaced with methionine, leucine andisoleucine. In another embodiment, the dioxygenase enzyme is modified,wherein the residue corresponding to position 362 of SEQ ID NO: 1 isreplaced with methionine, leucine and isoleucine. In another embodiment,the dioxygenase enzyme is modified, wherein the residue corresponding toposition 227 of SEQ ID NO: 1 is replaced with methionine, leucine andisoleucine. In another embodiment, the dioxygenase enzyme is modified,wherein the residue corresponding to position 252 of SEQ ID NO: 1 isreplaced with methionine, leucine and valine. In another embodiment, thedioxygenase enzyme is modified, wherein the residue corresponding toposition 361 of SEQ ID NO: 1 is replaced with threonine. In anotherembodiment, the dioxygenase enzyme is modified, wherein the residuecorresponding to position 224 of SEQ ID NO: 1 is replaced withmethionine, phenylalanine and tryptophan. In another embodiment, thedioxygenase enzyme is modified, wherein the residue corresponding toposition 289 of SEQ ID NO: 1 is replaced with methionine, leucine andisoleucine. In another embodiment, the dioxygenase enzyme is modified,wherein the residue corresponding to position 323 of SEQ ID NO: 1 isreplaced with tryptophan, tyrosine and isoleucine. In anotherembodiment, the dioxygenase enzyme is modified, wherein the residuecorresponding to position 367 of SEQ ID NO: 1 is replaced withmethionine, leucine, isoleucine and tryptophan. In another embodiment,the dioxygenase enzyme is modified, wherein the residue corresponding toposition 187 of SEQ ID NO: 1 is replaced with methionine, phenylalanineand tryptophan. In another embodiment, the dioxygenase enzyme ismodified, wherein the residue corresponding to position 241 of SEQ IDNO: 1 is replaced with methionine, phenylalanine and tryptophan. Inanother embodiment, the dioxygenase enzyme is modified, wherein theresidue corresponding to position 363 of SEQ ID NO: 1 is replaced withmethionine, isoleucine and valine. In another embodiment, thedioxygenase enzyme is modified, wherein the residue corresponding toposition 239 of SEQ ID NO: 1 is replaced with leucine. In anotherembodiment, the dioxygenase enzyme is modified, wherein the residuecorresponding to position 251 of SEQ ID NO: 1 is replaced withmethionine, isoleucine and proline. In another embodiment, thedioxygenase enzyme is modified, wherein the residue corresponding toposition 265 of SEQ ID NO: 1 is replaced with methionine, isoleucine andproline. In another embodiment, the dioxygenase enzyme is modified,wherein the residue corresponding to position 226 of SEQ ID NO: 1 isreplaced with methionine, valine, isoleucine and leucine. In anotherembodiment, the dioxygenase enzyme is modified, wherein the residuecorresponding to position 212 of SEQ ID NO: 1 is replaced withphenylalanine, leucine, isoleucine or tryptophan. In another embodiment,the dioxygenase enzyme is modified, wherein the residue corresponding toposition 217 of SEQ ID NO: 1 is replaced with methionine, isoleucine orleucine. In another embodiment, the dioxygenase enzyme is modified,wherein the residue corresponding to position 228 of SEQ ID NO: 1 isreplaced with methionine, isoleucine or leucine. In another embodiment,the dioxygenase enzyme is modified, wherein the residue corresponding toposition 210 of SEQ ID NO: 1 is replaced with leucine.

In some aspects, at least one of the substrate-specificity positionscorresponding to amino acids selected from the group consisting of A361,F336, F347, F364, F368, F371, G362, I227, I252, L224, L289, L323, L367,N187, N241, N363, P239, Q251, Q265, 5226, V212, V217, V228 and W210 ofSEQ ID NO: 1 has been replaced with one of the corresponding disclosedamino acids to alter the respective substrate-specificity residue.

In some other aspects, two or more of the substrate-specificitypositions corresponding to amino acids selected from the groupconsisting of A361, F336, F347, F364, F368, F371, G362, I227, I252,L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212,V217, V228 and W210 of SEQ ID NO: 1 have been replaced with one of thecorresponding disclosed amino acids to alter the respectivesubstrate-specificity residue.

In yet some other aspects, at least 3 and up to 24 of thesubstrate-specificity positions corresponding to amino acids selectedfrom the group consisting of A361, F336, F347, F364, F368, F371, G362,I227, I252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265,S226, V212, V217, V228 and W210 of SEQ ID NO: 1 have been replaced withone of the corresponding disclosed amino acids to alter the respectivesubstrate-specificity residue.

In some embodiments, the dioxygenase enzyme has been modified or mutatedto alter one or more one of the substrate-specificity residues. Incertain embodiments, the dioxygenase enzyme is modified, wherein theresidue corresponding to position 361 of SEQ ID NO: 6 is replaced with aresidue selected from methionine, leucine and isoleucine. In anotherembodiment, the dioxygenase enzyme is modified, wherein the residuecorresponding to position 336 of SEQ ID NO: 6 is replaced with leucine,methionine, isoleucine and tryptophan. In another embodiment, thedioxygenase enzyme is modified, wherein the residue corresponding toposition 347 of SEQ ID NO: 6 is replaced with tryptophan, tyrosine andisoleucine. In another embodiment, the dioxygenase enzyme is modified,wherein the residue corresponding to position 364 of SEQ ID NO: 6 isreplaced with methionine, alanine, isoleucine, leucine and tryptophan.In another embodiment, the dioxygenase enzyme is modified, wherein theresidue corresponding to position 368 of SEQ ID NO: 6 is replaced withtyrosine, tryptophan, leucine, isoleucine and methionine. In anotherembodiment, the dioxygenase enzyme is modified, wherein the residuecorresponding to position 371 of SEQ ID NO: 6 is replaced withmethionine, leucine and isoleucine. In another embodiment, thedioxygenase enzyme is modified, wherein the residue corresponding toposition 362 of SEQ ID NO: 6 is replaced with methionine, leucine andisoleucine. In another embodiment, the dioxygenase enzyme is modified,wherein the residue corresponding to position 227 of SEQ ID NO: 6 isreplaced with methionine, leucine and isoleucine. In another embodiment,the dioxygenase enzyme is modified, wherein the residue corresponding toposition 252 of SEQ ID NO: 6 is replaced with methionine, leucine andvaline. In another embodiment, the dioxygenase enzyme is modified,wherein the residue corresponding to position 361 of SEQ ID NO: 6 isreplaced with threonine. In another embodiment, the dioxygenase enzymeis modified, wherein the residue corresponding to position 224 of SEQ IDNO: 6 is replaced with methionine, phenylalanine and tryptophan. Inanother embodiment, the dioxygenase enzyme is modified, wherein theresidue corresponding to position 289 of SEQ ID NO: 6 is replaced withmethionine, leucine and isoleucine. In another embodiment, thedioxygenase enzyme is modified, wherein the residue corresponding toposition 323 of SEQ ID NO: 6 is replaced with tryptophan, tyrosine andisoleucine. In another embodiment, the dioxygenase enzyme is modified,wherein the residue corresponding to position 367 of SEQ ID NO: 6 isreplaced with methionine, leucine, isoleucine and tryptophan. In anotherembodiment, the dioxygenase enzyme is modified, wherein the residuecorresponding to position 187 of SEQ ID NO: 6 is replaced withmethionine, phenylalanine and tryptophan. In another embodiment, thedioxygenase enzyme is modified, wherein the residue corresponding toposition 241 of SEQ ID NO: 6 is replaced with methionine, phenylalanineand tryptophan. In another embodiment, the dioxygenase enzyme ismodified, wherein the residue corresponding to position 363 of SEQ IDNO: 6 is replaced with methionine, isoleucine and valine. In anotherembodiment, the dioxygenase enzyme is modified, wherein the residuecorresponding to position 239 of SEQ ID NO: 6 is replaced with leucine.In another embodiment, the dioxygenase enzyme is modified, wherein theresidue corresponding to position 251 of SEQ ID NO: 6 is replaced withmethionine, isoleucine and proline. In another embodiment, thedioxygenase enzyme is modified, wherein the residue corresponding toposition 265 of SEQ ID NO: 6 is replaced with methionine, isoleucine andproline. In another embodiment, the dioxygenase enzyme is modified,wherein the residue corresponding to position 226 of SEQ ID NO: 6 isreplaced with methionine, valine, isoleucine and leucine. In anotherembodiment, the dioxygenase enzyme is modified, wherein the residuecorresponding to position 212 of SEQ ID NO: 6 is replaced withphenylalanine, leucine, isoleucine or tryptophan. In another embodiment,the dioxygenase enzyme is modified, wherein the residue corresponding toposition 217 of SEQ ID NO: 6 is replaced with methionine, isoleucine orleucine. In another embodiment, the dioxygenase enzyme is modified,wherein the residue corresponding to position 228 of SEQ ID NO: 6 isreplaced with methionine, isoleucine or leucine. In another embodiment,the dioxygenase enzyme is modified, wherein the residue corresponding toposition 210 of SEQ ID NO: 6 is replaced with leucine.

In some aspects, at least one of the substrate-specificity positionscorresponding to amino acids selected from the group consisting of A361,F336, F347, F364, F368, F371, G362, I227, I252, L224, L289, L323, L367,N187, N241, N363, P239, Q251, Q265, 5226, V212, V217, V228 and W210 ofSEQ ID NO: 6 has been replaced with one of the corresponding disclosedamino acids to alter the respective substrate-specificity residue.

In some other aspects, two or more of the substrate-specificitypositions corresponding to amino acids selected from the groupconsisting of A361, F336, F347, F364, F368, F371, G362, I227, I252,L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, S226, V212,V217, V228 and W210 of SEQ ID NO: 6 have been replaced with one of thecorresponding disclosed amino acids to alter the respectivesubstrate-specificity residue.

In yet some other aspects, at least 3 and up to 24 of thesubstrate-specificity positions corresponding to amino acids selectedfrom the group consisting of A361, F336, F347, F364, F368, F371, G362,I227, I252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265,S226, V212, V217, V228 and W210 of SEQ ID NO: 6 have been replaced withone of the corresponding disclosed amino acids to alter the respectivesubstrate-specificity residue.

In an exemplary embodiment, the modified dioxygenase enzyme is derivedfrom a corresponding unmodified dioxygenase that is at least about 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or99.5% identical to a polypeptide selected from SEQ ID NOS: 1-8.

In some embodiments, the present disclosure relates to a polypeptidewith increased βHIV synthase activity, wherein the polypeptide sequenceis derived from Yarrowia lipolytica and is at least 65% identical to apolypeptide selected from either of SEQ ID NOs: 4-5 and has beenmodified or mutated to alter one or more one the substrate-specificityresidues. In certain embodiments, the polypeptide is modified at one ormore positions corresponding to amino acids selected from A374, F349,F360, F377, F381, I384, G375, V240, I265, A374, L237, I302, L336, L380,N200, N254, N377, P252, Q264, Q278, 5239, V225, 1230, V241 and W223. Inan exemplary embodiment, the modified decarboxylase enzyme is derivedfrom a corresponding unmodified decarboxylase that is at least about65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 99.5% identical to a polypeptide selected from either of SEQ IDNOs: 4-5.

Corresponding amino acids in other decarboxylases are easily identifiedby visual inspection of the amino acid sequences or by usingcommercially available homology software programs. Thus, given thedefined regions for changes and the assays described in the presentapplication, one with skill in the art can make one or a number ofmodifications which would result in an increased ability to specificallycatalyze the conversion of KIC to βHIV, in any homologous dioxygenaseenzyme of interest. The modified polypeptides can be optimally alignedwith the corresponding unmodified, wild-type dioxygenase enzymes togenerate a similarity score which is at least about 50%, more preferablyat least about 60%, more preferably at least about 70%, more preferablyat least about 80%, more preferably at least about 90%, or mostpreferably at least about 95% of the score for the reference sequenceusing the BLOSUM82 matrix, with a gap existence penalty of 11 and a gapextension penalty of 1.

In some embodiments, the non-natural microorganism expresses oroverexpresses a nucleic acid encoding fragments of the disclosedpolypeptides which comprises at least 25, 30, 40, 50, 100, 150, 200,250, 300 or 375 amino acids and retain βHIV synthase activity. Suchfragments may be obtained by deletion mutation, by recombinanttechniques that are routine and well-known in the art, or by enzymaticdigestion of the polypeptides of interest using any of a number ofwell-known proteolytic enzymes.

In some embodiments, the non-natural microorganism comprises at leastone nucleic acid molecule encoding a polypeptide with βHIV synthaseactivity, wherein said polypeptide is at least about 65% identical to apolypeptide selected from SEQ ID NOS: 1-148 Further within the scope ofpresent disclosure are recombinant microorganisms comprising at leastone nucleic acid molecule encoding a polypeptide with βHIV synthaseactivity, wherein said polypeptide is at least about 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical toa polypeptide selected from SEQ ID NOS: 1-149.

In accordance with the present disclosure, any number of mutations canbe made to the βHIV synthase enzymes, and in certain embodiments,multiple mutations can be made to result in an increased ability tocatalyze the conversion of KIC to βHIV with high catalytic efficiency.Such mutations can include point mutations, frame shift mutations,deletions, and insertions. In certain embodiments, one or more (e.g.,one, two, three, four, five, six, seven, eight, nine, ten or more, etc.)point mutations may be preferred.

In some embodiments, the βHIV synthase will have an intact C-terminus.As defined herein, the C-terminus of HPPD is the stretch of residuesthat include the C-terminal α-helix that shields the active site. Forexample, in SEQ ID NO: 1, the stretch of amino acids from 361 to 393 areconsidered the C-terminus. In some embodiments, the residues comprisingthe C-terminus are modified to allow increased activity with KIC. Insome embodiments, the C-terminus of the HPPD is in a conformation tohave the highest specificity for KIC.

In one embodiment, the βHIV metabolic pathway is localized to thecytosol of the non-natural microorganism. In one embodiment, thenon-natural microorganism comprises a βHIV metabolic pathway with atleast one pathway enzyme localized in the cytosol.

In some embodiments, the non-natural microorganism belongs to a genusselected from the group consisting of Escherichia, Corynebacterium,Lactobacillus, Lactococcus and Bacillus. In some embodiments, thenon-natural microorganism belongs to a genus selected from the groupconsisting of Saccharomyces, Kluyveromyces, Issatchenkia, Galactomyces,Pichia and Candida.

In some embodiments where the non-natural microorganism is a eukaryote,the βHIV metabolic pathway is expressed or overexpressed in its cytosol.

In certain embodiments, the non-natural microorganism comes in contactwith a carbon source in a fermenter to produce βHIV and introducing intothe fermenter sufficient nutrients where the final concentrationofβ-hydroxyisovalerate concentration in the fermentation broth isgreater than about 10 mg/L (for example, greater than about 100 mg/L,for example, greater than about 1 g/L, greater than about 5 g/L, greaterthan about 10 g/L, greater than about 20 g/L, greater than about 40 g/L,greater than 50 g/L), but usually below 150 g/L. In certain embodiments,the carbon source is selected from the group consisting of glucose,xylose, arabinose, sucrose, fructose, lactose, glycerol, and mixturesthereof.

In some embodiments, βHIV thus produced is optionally recovered from thefermentation broth by first removing the cells, followed by separatingthe aqueous phase from the clarified fermentation broth along with theother by-products of the fermentation. In some embodiments, the βHIV isco-purified with other fermentation-derived products, wherein thecomposition comprises at least one fermentation-derived impurity. Insome embodiments, fermentation-derived products are selected from thegroup consisting of organic acids and amino acids. In some embodiments,βHIV synthesized according to the present disclosure is substantiallydevoid of chloroform or hydrochloric acid.

The object of the present disclosure is further illustrated by thefollowing examples that should not be construed as limiting. Examplesare provided for clarity of understanding. While the object of thepresent disclosure has been described in connection with embodimentsthereof, it will be understood that it is capable of furthermodifications and this disclosure is intended to cover variations, useror adaptations of the present disclosure following, in general, theprinciples of the present disclosure and including such departures fromthe present disclosure as come within known or customary practice withinthe art to which the present disclosure pertains and as may be appliedto the essential features hereinbefore set forth and as follows in thescope of the appended claims. The contents of all references, patents,and published patent applications cited throughout this application, aswell as the Figures and the Sequence Listings, are incorporated hereinby reference for all purposes.

EXAMPLES Example 1: Selection of Microorganisms

This example illustrates a method to select microorganisms that are apriori suited to produce βHIV. Several bacteria and yeasts were testedfor their ability to grow in the presence of βHIV. Bacterialstrains—Corynebacterium glutamicum NRRL B-2784, Escherichia coli MG1655and yeast strains—Saccharomyces cerevisiae CENPK.2, Kluyveromycesmarxianus NRRL Y-318, NRRL Y-6373, Pichia kudriavzevii NRRL Y-7551, NRRLY-5396 were selected as exemplary microorganism to evaluate theirability to grow in the presence of βHIV. Growth is considered anindicator of overall metabolic activity. The bacterial strains werestarted in LB medium and the yeast strains were started in YPD medium atconditions that are reportedly ideal for optimal growth. For example, E.coli strain was started from cryo-vials in LB medium at 37° C. and S.cerevisiae was started from cryo-vials in YPD medium at 30° C.

Seed cultures of all the strains were transferred into minimal mineralsalts medium, supplemented with 2% glucose. To the same media βHIV wasadded to a final concentration of 20 g/L, 40 g/L or 50 g/L. Growth ofthe microorganisms in the presence ofβHIV was monitored in BioLector II(m2p Labs, Aachen, Germany) in triplicate. The maximum specific growthrate of these microorganisms in the presence of various concentrationsof βHIV was calculated and shown in FIG. 3. Typically yeasts grew fasterthan bacteria at higher concentrations of βHIV, suggesting fasterglucose uptake. Specifically, Y-6373, Y-7551 and Y-5396 exhibited thefastest growth and are potential candidates to host the βHIV metabolicpathway. This example illustrates a method to screen and selectappropriate host microorganisms for producing βHIV.

Example 2: Production of βHIV in Bacteria

This example illustrates the production of βHIV in bacteria and yeast. Astrain of Corynebacterium glutamicum MV-KICF1 (Applied Microbiol, MicrobBiotech ,8, 351-360) that was metabolically modified to produceα-ketoisocaproate was used to introduce a gene that encodes for apolypeptide encoding SEQ ID NO: 1. The codons in the nucleic acidsequence of the gene were optimized according to the codon usage of thebacterium and the DNA was cloned into pZ8-ptac vector (Cleto et al., ACSSynth Biol. 2016 May 20; 5(5): 375-385) and transformed into MV-KICF1 byelectroporation. The non-natural bacterial strain endowed with thecapability to produce βHIV was propagated in Brain-Heart Infusion mediumand cultivated in CGXII medium (Hoffman et al., J Appl Microbiol., 2014,117: 663-678) using glucose as the main carbon source to evaluate theβHIV production. Substrate consumption and product formation wasevaluated on Agilent 1200 HPLC using 5mM H2SO4 as the mobile phase withAminex HPX-87c column (BioRad, Hercules, CA). As illustrated in FIG. 4,the non-natural bacterium comprising βHIV synthase produced more βHIVthan its parent wild-type.

Example 3: Assembling βHIV Metabolic Pathway in Yeast Cytosol

This example provides methods to assemble βHIV metabolic pathway shownin FIG. 1 in Y-5396 yeast. The example also provides methods fordiscriminating between enzymes (genes) that perform the same function.All DNA modifications were performed based on a reference genome that isavailable publicly (Douglass et al., 2018, PLoS Pathog, 14(7):e1007138).To facilitate use of URA3 as a selection marker, both alleles of URA3 inthe diploid yeast Y-5396 were deleted. The first URA3 allele wasreplaced by a KanMX cassette conferring Geneticin resistance and flankedby loxP sites by transforming a KanMX cassette flanked by ˜500 bases ofhomology upstream and downstream of URA3 into Y-5396. Transformants wereselected on media containing Geneticin and insertion of the cassette inthe URA3 locus confirmed by PCR, creating strain SB500. To delete thesecond URA3 allele ˜500 bases upstream and ˜500 bases downstream of URA3were joined via overlap extension PCR to create a clean deletionconstruct and transformed into SB500. Transformants were plated on5-fluorootic acid to select for mutants that had lost both alleles ofURA3. The clean deletion of the second allele was confirmed by PCR andthe KanMX cassette insertion was re-confirmed, to create strain SB501.Finally, the KanMX cassette was removed by transient expression of Crerecombinase from an unstable plasmid that conferred resistance toHygromycin. Colonies were then screened for loss of Geneticin resistance(loss of KanMX), loss of Hygromycin resistance (plasmid marker), andability to grow on 5-FOA (double deletion of URA3). Genomic DNA at theURA3 locus was PCR-amplified and sequenced to confirm the deletions hadoccurred as expected. The resulting strain called SB502, which has thetwo copies of the URA3 gene deleted to facilitate the use of uracil forselection.

Genes encoding for enzymes in the βHIV metabolic pathway were insertedin the chromosome at intergenic loci using homologous recombination.Chromosomal integration of heterologous genes to assemble the βHIVmetabolic pathway is illustrated for α-isopropylmalate synthase (IPMS)as an example. One plasmid (p1) was constructed containing theintegration site 5′ homology arm, IPMS expression cassette, and the 3′two-thirds of the URA3 cassette. A second plasmid (p2) was constructedwith the 5′ two-thirds of the URA3 cassette (such that there was ˜500bases overlap with p1) and the integration site 3′ homology arm. Thisstrategy decreases the rate of unwanted recombination events becauseonly when both halves integrate into the same site will a functionalURA3 cassette be formed. All PCR reactions were performed using NEB Q5high fidelity polymerase according to manufacturer's instructions.Plasmids were assembled using NEBuilder HiFi assembly mix according tomanufacturer's instructions, routinely using 30 base pair overlaps tofacilitate assembly. The URA3 cassette is flanked by loxP sites tofacilitate removal of the marker by expression of Cre recombinase. Genesencoding for enzymes with SEQ ID NOS: 308-312 were evaluated for theirIPMS activity.

Plasmids shown in Table 1 were assembled using NEBuilder HiFi assemblymix according to manufacturer's instructions, routinely using 30 basepair overlaps to facilitate assembly. IPMS genes were inserted into twoseparate intergenic loci on chromosome A (NC_042506). Intergenic locusA2193833 (aka igA2.2) and A1207782 (aka igA1.2).

TABLE 1 Relevant plasmids used to modify strains in this example PlasmidUse Genotype/relevant genes pSB011 Insert IPMS cassette and ura3 marker5′ HA igA2.2, ioTDH3p-SEQ ID NO: into io intergenic locus igA2.2308-ioTKLt; lox66-ioTALt-ura3 3′ pSB012 Insert IPMS cassette and ura3marker 5′ HA igA2.2, ioTDH3p-SEQ ID NO: into io intergenic locus igA2.2309-ioTKLt; lox66-ioTALt-ura3 3′v pSB013 Insert IPMS cassette and ura3marker 5′ HA igA2.2, ioTDH3p-SEQ ID NO: into io intergenic locus igA2.2310-ioTKLt; lox66-ioTALt-ura3 3′v pSB014 Insert IPMS cassette and ura3marker 5′ HA igA2.2, ioTDH3p-SEQ ID NO: into io intergenic locus igA2.2311-ioTKLt; lox66-ioTALt-ura3 3′ pSB015 Insert IPMS cassette and ura3marker 5′ HA igA2.2, ioTDH3p-SEQ ID NO: into io intergenic locus igA2.2312-ioTKLt; lox66-ioTALt-ura3 3′v pSB017 Insert IPMS cassette and ura3marker Ura3 5′-ioPGKp-lox71, into io intergenic locus igA2.2 3′ HAigA2.2 pSB019 Insert IPMS cassette and ura3 marker 5′ HA igA1.2,ioTDH3p- into io intergenic locus igA1.2 C. glutamicum leuA B018-ioTKLt;lox66- ioTALt-ura3 3′ pSB020 Insert IPMS cassette and ura3 marker 5′ HAigA1.2, ioTDH3p- into io intergenic locus igA1.2 C. glutamicum leuACP-ioTKLt; lox66- ioTALt-ura3 3′ pSB021 Insert IPMS cassette and ura3marker Ura3 5′-ioPGKp-lox71, into io intergenic locus igA1.2 3′ HAigA1.2 pEC010 Express Cre recombinase in io strains ioPGKp-cre-CYC1t;KanMX, ioCEN0.8, ioARS

To construct pSB011-15 the 5′ homology arm and ioTDH3 promoter were PCRamplified with primers shown in SEQ ID NO: 321 and SEQ ID NO: 322 andSEQ ID NO: 323 and SEQ ID NO: 324 respectively, using SB502 genomic DNAas template. The IPMS genes were codon optimized for Issatchenkiaorientalis and synthesized as gene fragments by Twist Biosciences (SanFrancisco, Calif.). The genes needed to be split into two fragmentsbecause of their length and complexity. The ioTKL terminator & 3′portion of the URA3 cassette and vector backbone (pTwist-Kan high copy),were PCR amplified from a plasmid synthesized by Twist Biosciences. Toconstruct pSB017 the 3′ homology arm was amplified using primers SEQ IDNO: 325 and SEQ ID NO: 326 and SB502 genomic DNA as template. The 5′portion of the URA3 cassette and vector backbone was amplified usingprimers ig2.2p2 gib vec F+R and plasmid ig1.6p2 as template. Clones werescreened by PCR and/or restriction digest for proper assembly andsequences were confirmed via Sanger sequencing. The p1 and p2 insertswere liberated from their vector backbones via restriction digest andinserts purified via gel extraction (NEB Monarch gel purification kit)to be transformed into suitable yeast strain.

All transformations were performed using the lithium acetate method asdescribed in Geitz & Schiestl, 2007, Nature Protocols, 31-34. Individualtransformants were screened using colony PCR to confirm correctintegration of the 5′ flank (using primers SEQ ID NO: 327 and SEQ ID NO:328) and 3′ flank (using primers SEQ ID NO: 329 and SEQ ID NO: 330) andto confirm correct assembly of the ura3 marker (using primers SEQ ID NO:331 and SEQ ID NO: 332) and presence of the gene of interest (SEQ ID NO:333 and SEQ ID NO: 334). Primers amplifying the native integration site(SEQ ID NO: 327 and SEQ ID NO: 329) were also used to identify anyheterozygosity. The resulting strains containing a single copy of a geneencoding enzymes with SED ID NOs: 308-312, designated SB507-SB511,respectively, were assayed for IPMS activity. The activity wasdetermined by measuring the amount of free CoA liberated as described inKohlhaw and Leary, 1969, Vol. 244, No. 8 pp. 2218-2225. Total proteinconcentration in cell lysates was measured using Bradford assay. Therecorded activity from these strains is expressed in nmol/mg prot/minand shown in Table 2.

TABLE 2 Enzyme activity in yeast strains Strain Sequence Activity SB507SEQ ID NO: 308 86.5 SB508 SEQ ID NO: 309 56.0 SB509 SEQ ID NO: 310 25.5SB510 SEQ ID NO: 311 89.3 SB511 SEQ ID NO: 312 5.4 SB512 Control 5.5

As illustrated in Table 2, even a single copy of the gene couldsignificantly enhance enzyme activity. Strains SB507 and SB510 wereselected for inserting a second copy of the gene to make the locushomozygous. These strains were transformed with 1 μg of pEC010 plasmidcontaining a Cre expression cassette and KanMX cassette conferringresistance to Geneticin. Transformants were plated on YPD+500 ug/mLG418-sulfate. A single colony was used to inoculate YPD broth +500 ug/mLG418-sulfate and grown overnight. The G418 culture was then used toinoculate SC+1 g/L 5-FOA which selected for clones that had lost theura3 marker. The 5-FOA culture was grown for 24-48 hours until visiblegrowth was observed then cells were streaked for isolation onto a YPDplate. Single colonies were replica plated onto YPD, YPD+G418500 andSC-Ura plates. Clones that did not grow in the presence of G418 (hadlost pEC010) or SC-Ura (lacked URA3) were screened via colony PCR toconfirm URA3 loop-out. The second copy of the gene was integrated in asecond round of transformation using the same p 1 and p2 constructs.Successful integration and homozygosity were confirmed using colony PCR.The resulting strain was rendered auxotrophic for uracil by repeatingthe method described above, to facilitate further modification. In themanner described above, the other genes in the βHIV metabolic pathwayshown in FIG. 1 were inserted subsequently. Sequences of exemplaryenzymes that catalyze various steps of the βHIV metabolic pathway arestep (b): SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300step (c): SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, step (d): SEQID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, step (f):SEQ ID NO: 314, SEQ ID NO: 315 and step (g): SEQ ID NO: 316, SEQ ID NO:317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320. Therefore, theresulting yeast strains have the multiple variations of the βHIVmetabolic pathway leading up to KIC.

A strain, designated SB553, comprising homozygous integration of genesthat encode for SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 304, SEQ IDNO: 308, SEQ ID NO: 315 and SEQ ID NO: 319, was grown in 50 mL of yeastminimal salts medium supplemented with 40 mg/L of uracil, trace metals,vitamins and glucose as the carbon source (Verduyn, et al. Yeast 8, 7:501-517, 1992). After 44 h of growth, the yeast culture was harvestedand centrifuged to remove the yeast cells. The clarified supernatantswere analyzed for residual glucose and βHIV synthesis via HPLC asdescribed in Example 2. The strain SB502 was also grown under identicalconditions to serve as a control. SB502 produced only 0.32 g/L of KICwhile SB553 produced 1.84 g/L of KIC. Increased KIC production isclearly illustrative of the increased activity of all the enzymesexpressed or overexpressed in the yeast cytosol.

Example 4: Production of βHIV in Yeast

This example will illustrate the integration of the complete βHIVmetabolic pathway that will result in βHIV production by a non-naturalyeast. All genetic manipulations were carried out using the methodsdescribed in Example 3. Step (a) of the βHIV metabolic pathway shown inFIG. 1, encoding for βHIV synthase, was assembled in strain SB553.Codon-optimized sequences of genes that encode for βHIV synthasevariants corresponding to SEQ ID NO: 1 and SEQ ID NO: 335 werechromosomally integrated in SB553 to result in strains SB556 and SB557.The three strains were grown under identical conditions using minimalsalts medium described in Example 3. After 42 h of growth, the culturewas harvested, and cells separated by centrifugation. The clarifiedsupernatant was analyzed for βHIV using an HPLC as described in Example3. As illustrated in FIG. 5, supernatant from SB553 did not have anydetectable βHIV. However, SB556 supernatant had 0.04 g/L ofβHIV andSB557 supernatant had 0.12 g/L ofβHIV. The result confirms theconversion of glucose into βHIV in SB556 and SB557 yeast using themetabolic pathway shown in FIG. 1 and the ability of the yeast cell toexport βHIV into the medium using a transporter. Furthermore, the resultalso illustrates increased βHIV production using different variantsofβHIV.

Various embodiments of systems, devices, and methods have been describedherein. These embodiments are given only by way of example and are notintended to limit the scope of the claimed inventions. It should beappreciated, moreover, that the various features of the embodiments thathave been described may be combined in various ways to produce numerousadditional embodiments. Moreover, while various materials, dimensions,shapes, configurations and locations, etc. have been described for usewith disclosed embodiments, others besides those disclosed may beutilized without exceeding the scope of the claimed inventions.

Persons of ordinary skill in the relevant arts will recognize that thesubject matter hereof may comprise fewer features than illustrated inany individual embodiment described above. The embodiments describedherein are not meant to be an exhaustive presentation of the ways inwhich the various features of the subject matter hereof may be combined.Accordingly, the embodiments are not mutually exclusive combinations offeatures; rather, the various embodiments can comprise a combination ofdifferent individual features selected from different individualembodiments, as understood by persons of ordinary skill in the art.Moreover, elements described with respect to one embodiment can beimplemented in other embodiments even when not described in suchembodiments unless otherwise noted.

Although a dependent claim may refer in the claims to a specificcombination with one or more other claims, other embodiments can alsoinclude a combination of the dependent claim with the subject matter ofeach other dependent claim or a combination of one or more features withother dependent or independent claims. Such combinations are proposedherein unless it is stated that a specific combination is not intended.

Any incorporation by reference of documents above is limited such thatno subject matter is incorporated that is contrary to the explicitdisclosure herein. Any incorporation by reference of documents above isfurther limited such that no claims included in the documents areincorporated by reference herein. Any incorporation by reference ofdocuments above is yet further limited such that any definitionsprovided in the documents are not incorporated by reference hereinunless expressly included herein.

For purposes of interpreting the claims, it is expressly intended thatthe provisions of 35 U.S.C. § 112(f) are not to be invoked unless thespecific terms “means for” or “step for” are recited in a claim.

What is claimed is:
 1. A non-natural microorganism capable of producingbeta-hydroxyisovalerate (βHIV), wherein the non-natural microorganism ismodified to provide more βHIV than the wild-type parent.
 2. Thenon-natural microorganism of claim 1, wherein the non-naturalmicroorganism comprises a metabolic pathway for producing βHIV.
 3. Thenon-natural microorganism of claim 2, wherein the metabolic pathwaycomprises one or more steps of (i) pyruvate to acetolactate, (ii)acetolactate to 2,3-dihydroxyisovalerate, (iii) 2,3-dihydroxyisovalerateto α-ketoisovalerate, (iv) α-ketoisovalerate to α-isopropylmalate, (v)α-isopropylmalate to β-isopropylmalate, (vi) β-isopropylmalate toα-ketoisocaproate and (vii) α-ketoisocaproate to βHIV.
 4. Thenon-natural microorganism of claim 2, wherein the metabolic pathwaycomprises one or more steps of (i) pyruvate into acetolactate, (ii)acetolactate into 2,3-dihydroxyisovalerate, (iii)2,3-dihydroxyisovalerate into α-ketoisovalerate, (iv) α-ketoisovalerateinto 2-isopropylmalate, (v) 2-i sopropylmalate into 2-i sopropylmaleate,(vi) 2-i sopropylmaleate into 3-i sopropylmalate, (vii) 3-isopropylmalate into 2-i sopropyl-3-oxosuccinate, (viii)2-isopropyl-3-oxosuccinate into α-ketoisocaproate, and (ix)α-ketoisocaproate into βHIV.
 5. The non-natural microorganism of claim2, wherein the non-natural microorganism is a eukaryote, and wherein themetabolic pathway hosts at least one βHIV pathway enzyme selected fromthe group consisting of acetolactate synthase having at least 80%identity to the group consisting of SEQ ID NOs: 297-300, cytosolicketo-acid reductoisomerase having at least 80% identity to the groupconsisting of SEQ ID NOs: 301-303, cytosolic dihydroxyacid dehydratasehaving at least 80% identity to the group consisting of SEQ ID NOs:304-307, cytosolic 2-isopropylmalate synthase having at least 80%identity to the group consisting of SEQ ID NOs: 308-313, cytosolicisopropylmalate isomerase having at least 80% identity to the groupconsisting of SEQ ID NOs: 314-315, cytosolic 3-isopropylmalatedehydrogenase having at least 80% identity to the group consisting ofSEQ ID NOs: 316-320, and cytosolic βHIV synthase having at least 65%identity to the group consisting of SEQ ID NOS: 1-148.
 6. Thenon-natural microorganism of claim 1, wherein the non-naturalmicroorganism comprises at least one nucleic acid encoding a polypeptidewith beta hydroxyisovalerate synthase activity, wherein said polypeptideis at least about 65% identical to at least one polypeptide selectedfrom the group of SEQ ID NOS: 1-148.
 7. The non-natural microorganism ofclaim 1, wherein the non-natural microorganism comprises at least onenucleic acid encoding a polypeptide with beta hydroxyisovaleratesynthase activity, wherein said polypeptide is at least about 65identical to at least one polypeptide selected from SEQ ID NO: 1 or SEQID NO:
 6. 8. The non-natural microorganism of claim 1, wherein thenon-natural microorganism comprises at least one nucleic acid encoding apolypeptide with βHIV synthase activity derived from the groupconsisting of Rattus norvegicus, Yarrowia lipolytica, and Homo sapiens.9. The non-natural microorganism of claim 1, wherein the non-naturalmicroorganism comprises a non-natural enzyme, wherein the non-naturalenzyme has been modified or mutated to increase the ability of theenzyme to preferentially utilize α-ketoisocaproate as its substrate, andwherein the non-natural enzyme comprises one or more modifications ormutations at substrate-specificity positions corresponding to aminoacids selected from A361, F336, F347, F364, F368, F371, G362, I227,I252, L224, L289, L323, L367, N187, N241, N363, P239, Q251, Q265, 5226,V212, V217, V228 and W210 of any of SEQ ID NO: 1 or SEQ ID NO:
 6. 10.The non-natural microorganism of claim 1, wherein the non-naturalmicroorganism comprises a non-natural enzyme, wherein the non-naturalenzyme comprises one or more modifications or mutations atsubstrate-specificity positions selected from the group consisting ofleucine, isoleucine or methionine at position 361, leucine, isoleucine,methionine or tryptophan at position 336, tryptophan, tyrosine orisoleucine at position 347, alanine, leucine, isoleucine, methionine ortryptophan at position 364, tyrosine, tryptophan, leucine, isoleucine ormethionine at position 368, leucine, isoleucine or methionine atposition 371, leucine, isoleucine or methionine at position 362,leucine, valine or methionine at position 227, leucine, valine ormethionine at position 252, phenylalanine, tryptophan or methionine atposition 224, leucine, valine or methionine at position 289, tryptophan,tyrosine or isoleucine at position 323, leucine, isoleucine, tryptophanor methionine at position 367, phenylalanine, tryptophan or methionineat position 187, phenylalanine, tryptophan or methionine at position241, isoleucine, methionine or valine at position 363, leucine atposition 239, methionine, isoleucine or proline at position 251,methionine, isoleucine or proline at position 265, valine, methionine,isoleucine or leucine at position 226, phenylalanine, leucine,isoleucine or tryptophan at position 212, isoleucine, leucine ormethionine at position 217, isoleucine, leucine or methionine atposition 228, and leucine at position 210 of SEQ ID NO: 1 or SEQ ID NO:6.
 11. The non-natural microorganism of claim 1, wherein the non-naturalmicroorganism is a prokaryotic microorganism or an eukaryoticmicroorganism.
 12. The non-natural microorganism of claim 1, wherein thenon-natural microorganism comprises a yeast or a bacteria.
 13. Thenon-natural microorganism of claim 12, wherein the non-naturalmicroorganism is a yeast selected from the group consisting ofSaccharomyces, Kluyveromyces, Pichia, Issatchenkia, Hansenula, andCandida.
 14. The non-natural microorganism of claim 12, wherein thenon-natural microorganism is Gram-positive bacteria or a Gram-negativebacteria, the Gram-positive bacteria selected from the group comprisingof Corynebacterium, Lactobacillus, Lactococcus and Bacillus, and theGram-negative bacteria selected from the group comprising of Escherichiaand Pseudomonas.
 15. The non-natural microorganism of claim 1, whereinthe non-natural microorganism is cultivated in a culture mediumcontaining a feedstock of a carbon source to produce βHIV.
 16. Thenon-natural microorganism of claim 1, wherein the microorganism iscultivated in a culture medium containing a feedstock of a carbon sourceto produce βHIV at a yield of at least about 0.1 percent up to 100percent of theoretical yield.
 17. The non-natural microorganism of claim1, wherein the microorganism is in contact with a carbon source in afermenter to produce βHIV, wherein the fermenter introduces sufficientnutrients such that a final βHIV concentration in the fermentation brothis greater than about 10 mg/L.
 18. The non-natural microorganism ofclaim 1, wherein the microorganism is in contact with a carbon source ina fermenter to produce βHIV, wherein the carbon source is selected fromthe group consisting of glucose, xylose, arabinose, sucrose, fructose,lactose, glycerol, and mixtures thereof.
 19. The non-naturalmicroorganism of claim 1, wherein the microorganism is in contact with acarbon source in a fermenter to produce a fermentation-derivedcomposition comprising βHIV and at least one fermentation derivedimpurity, wherein the at least one fermentation derived impuritypreferably comprises one or more amino acids and/or organic acids. 20.The non-natural microorganism of claim 1, wherein the microorganism isin contact with a carbon source in a fermenter to produce afermentation-derived composition comprising βHIV and at least onefermentation derived impurity, wherein the fermentation-derivedcomposition is devoid of any halogen-containing components.
 21. A methodof producing βHIV using a non-natural microorganism, the methodcomprising: culturing a non-natural microorganism in the presence of atleast one carbon source to produce a fermentation-derived compositioncomprising βHIV, wherein the non-natural microorganism is modified toprovide more βHIV than the wild-type parent; and isolating βHIV from thefermentation-derived composition.
 22. The method of claim 21, whereinthe fermentation-derived composition further comprises at least onefermentation derived component, and wherein the fermentation-derivedcomposition comprising βHIV prior to isolation is substantially devoidof a chemically derived inorganic residue chosen from chloroform,hydrochloric acid, and a halogen derivative.
 23. A compositioncomprising: βHIV produced by a non-natural microorganism, wherein theβHIV prior to any isolation or purification process has not been insubstantial contact with any halogen-containing component, thehalogen-containing component comprising hydrochloric acid or chloroform.